Deazaflavin Inhibitors of TDP2 with Cellular Activity Can Affect Etoposide Influx and/or Efflux.

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 "poisons". Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle.

Monepantel (MNP) is a new amino-acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO2 ) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 µm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin-monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 µm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well-known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO2 was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO2 was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO2 in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO2 production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO2 in liver microsomes of both sheep (58 to 98%, in a dose-dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO2 with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.

The effect of caffeine on peripheral vascular resistance in isolated perfused guinea pig hind limbs.

BACKGROUND: The role of caffeine in cardiovascular disease is controversial. Most of its pharmacologic actions are attributed to its role as an adenosine antagonist. Adenosine is one of the most important endogenous vasodilatative substances and is released under ischemic conditions, for example, in the skeletal muscle of patients with peripheral arterial occlusive disease. We aimed to investigate the influence of caffeine on peripheral vascular resistance and on the beneficial vasodilatory effect of adenosine in isolated perfused guinea pig hind limbs. MATERIALS AND METHODS: (1) Caffeine was administered at 0.5, 5, and 50 micromol/L under normoxic conditions. (2) The vasculature of the perfused guinea pig hind limb was precontracted with noradrenaline (3 micromol/L), followed by adenosine (10 micromol/L) under normoxic conditions. When vascular resistance (VR) had reached a steady state, caffeine was administered additionally at dosages of 0.5, 5, and 50 micromol/L. (3) This protocol was repeated using iloprost 0.1 micromol/L instead of adenosine as vasodilatory substance. (4) Under hypoxia, caffeine was again administered at the above dosages. (5) Under hypoxia, experiments with adenosine A2-receptor antagonists (alloxazine 10 micromol/L and ZM 241385 100 nmol/L) were done. RESULTS: Under normoxic conditions, 0.5 and 5 micromol/L caffeine had nearly no effect on vascular resistance compared with baseline conditions. A slight, but statistically not significant decrease in VR was achieved with 50 micromol/L caffeine. In the presence of noradrenaline, the vasodilatory effect of adenosine was reduced by 7.6 +/- 1.6% after the addition of 0.5 micromol/L caffeine, and by 37.3 +/- 3.8% at a dosage of 5 micromol/L caffeine. A dosage of 50 micromol/L caffeine completely abolished the vasodilatative effect of adenosine. In the presence of iloprost, only a slight but statistically insignificant inhibitory influence (0.9%) of caffeine at a dosage of 50 micromol/L could be seen. Hypoxia significantly reduced VR. Caffeine at 0.5 micromol/L diminished this effect by about 53.2 +/- 4.6% and abolished it at 5 and 50 micromol/L. The hypoxia-induced adenosine-mediated vasodilatation seems to be an adenosine A2A-receptor-mediated effect. CONCLUSIONS: The observed effect of hypoxia-induced vasodilatation in peripheral arteries may be the result of the vasodilatory effect of elevated endogenous adenosine during hypoxia. For patients with peripheral arterial disease, drinking of caffeine-containing beverages may reduce the beneficial vasodilatory effect of elevated endogenous adenosine levels.

Photochemical and pharmacokinetic properties of selected flavins.

Some photochemical and pharmacokinetic properties of riboflavin, lumiflavin and the 2',3',4',5' tetraacetyl, tetrapropionyl, tetrabutiryl and tetrapalmitoyl esters of riboflavin have been studied. The esters are considerably more photostable than riboflavin but less so than lumiflavin, and appear to be photosensitizers with a behaviour similar to that of riboflavin, promoting photoreactions of biological targets even in the absence of molecular oxygen. The various flavins display important differences in their pharmacokinetic behaviour. Riboflavin, lumiflavin and the short-chain esters (the acetyl and propionyl esters) are rapidly cleared from serum, and recovered in comparable amounts from the liver and kidneys. These results are in agreement with their hydrophilic or moderately hydrophobic character. In contrast, the longer-chain butiryl and palmitoyl esters exhibit a prolonged retention in serum and undergo a significantly larger accumulation in the liver as compared with the kidneys; they are also found in the spleen. In all cases the tissue uptake of these esters becomes appreciable only after 24 h. These results are coherent with the highly hydrophobic character of these esters, which induce a slow release from serum lipoproteins and have a preferential clearance via the bile-gut pathway, showing affinity for the components of the reticuloendothelial system. These long-chain riboflavin esters will probably have a greater and more persistent risk of photoinduced hepatotoxicity than riboflavin, lumiflavin and the short-chain esters.

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues.

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.

Smectite interactions with flavomononucleotide.

Adsorption isotherms and UV-visible and Mossbauer spectroscopic data point to specific interactions between flavomononucleotide (FMN) and Fe(3+)-smectite. The maximum amount of FMN adsorption was 0.3 mmole/g of Fe(3+)-smectite giving a 1:1 molar proportion of Fe3+ and FMN. The results suggest a Fe(3+)-FMN complex residing at the smectite surface. Other homoionic smectites (Cu2+, Zn2+, and Ca2+) exhibited lower levels of adsorption and less apparent specific interaction.